altered immune function in space: implications of a ... · well, and downstream tumor necrosis...

28

Sejiro Littleton and Dylan LudwickDavidson College

Faculty Mentor: Sophia SarafovaDavidson College

Altered Immune Function in Space: Implications of a Gravity Sensitive

Cytoskeleton

ABSTRACTFuture plans in space exploration have outlined missions to both asteroids and planets such as Mars. Extended spaceflight has long been associated with immunosuppressive effects. A grow-ing body of evidence suggests that microgravity plays an important role in astronaut immune dysfunction through its interactions with the cytoskeleton. This review summarizes the current understanding of the effects of microgravity on both adaptive and innate immunity, while dis-cussing the implications of cytoskeletal alterations in the search of a primary mechanism for gravity sensing. We conclude by addressing the need for more thorough investigation in order to fully understand the long-term consequences of cellular adaptation to low gravity conditions during extended spaceflight.

Explorations |Biological Sciences

Muscle atrophy and loss of bone density are among the topics best known by the public and scientists. However, serious concerns have been raised about impaired eyesight, psychological and physical stress, ionizing radiation, as well as latent viral reactivation and alterations to the immune system. The first immunologically relevant concerns were documented by the Apollo missions, which reported symptoms of “infectious illness” in 50% - 60% of crewmembers (1). To mini-mize this health risk NASA implemented the Health Stabilization Program starting with the Apollo 14 mission. This program significantly reduced the frequency of symp-toms by enforcing light quarantine on crew-members prior to launch. A summary of post flight debriefs in 106 shuttle missions reports 29 incidents of infectious disease out of 742

The technical limitations of launch, navi-gation and safe landing are not the only obstacles for placing humans in orbit or on celestial bodies. Research on space-faring hu-mans, cell cultures, and model organisms has shown that there are a great number of physi-ological changes that accompany this event.

Since the beginning of time humans have gazed unto the heavens with curiosity

and awe. In ancient times the stars guided farmers to the seasons and sailors across oceans. More recently, we have been inspired to take the first steps in exploration of the fi-nal frontier. We have sent a rover to Mars, landed the Philae probe on an asteroid, and for the past 15 years, through international cooperation, have maintained the orbital re-search lab known as the International Space Station (ISS).

1

29

crewmembers. Among those reported were fever and chills, fungal infections, flu-like symptoms, urinary tract infections, and viral diseases (1). Despite improvements, consis-tent reports of infection still remain, suggest-ing that there might be a separate effect of space flight directly on immune function.

If humankind is to conduct further missions and extended manned flights such as those required to place a human on Mars, we must understand the changes and risks imposed by microgravity and extended spaceflight. The focus of this paper is on the changes seen in the immune system under these circum-stances. We begin by laying out the relevant changes found in the T and B lymphocytes of the adaptive immune system. We move on to discuss the innate immune system and altera-tions that occur in Natural Killer (NK) cells, the third lymphocyte lineage, and monocytes and macrophages of the myeloid lineage.

The mechanism by which microgravity alters immune cell function remains unclear. We hypothesize that the cytoskeleton and its associated proteins are gravity sensors of the cell, and changes to their regulation induced by microgravity disrupt processes such as motility, signaling, gene expression, and cell proliferation. Given that cells of the immune system use their cytoskeleton to undergo cell division during proliferation, to alter their shape and migrate more than any other cell in the body through the processes of extravasa-tion, and to differentiate in response to vari-ous environmental stimuli, it stands to reason that the loss of Earth’s gravity could lead to dysfunction in the innate and adaptive im-mune responses.

Understanding these changes and the mechanisms which lead to altered immune function will be important to define the health risk of missions to Mars, other manned missions seeking to explore our solar system, and any prolonged time spent inhabiting an altered gravity environment such as the ISS. Additionally, this knowledge will bring a greater understanding to the possibility and nature of life on other planets, the structure and role of the cytoskeleton, and impart new

knowledge for application in treating disease on Earth.

ADAPTIVE IMMUNITY

T Lymphocytes

The inhibition of T lymphocyte activation by microgravity is a widely documented phe-nomenon dating back to the 1970s as initial observations from US and Soviet space mis-sions noted a reduced response to mitogen stimulation in the T cells of crew members upon returning to Earth (2). Subsequent ex-perimentation over the next two decades in Spacelab D1, SLS-1 (Space Launch System), and IML-2 (International Microgravity Laboratory) would go on to independently confirm the consistent and substantial disrup-tion of T cell activation in microgravity with 1g-onboard centrifuge controls (3, 4, 5).

The non-responsiveness of T lymphocytes may be attributed to disruption at one or more of the 3 signals involved in full activa-tion. The first of these, signal 1, is initiated through the specific binding of the T Cell Receptor (TCR) with a unique antigenic pep-tide presented on a Major Histocompatibility Complex (MHC) molecule by an Antigen Presenting Cell (APC). Signal 2, also known as costimulation, involves the triggering of CD28 on the T cell by CD80/86 on the APC. Signal 3 is a cytokine signal usually deliv-ered by Interleukin-2 (IL-2) in an autocrine fashion, binding to IL-2 Receptor (IL-2R) on the cell surface of T cells. If a cell should receive only signal 1 through its TCR, in other words there is no costimulation through signals 2 or 3, the cell will be rendered non-responsive to antigen. This process is known as anergy and it plays an important role in maintaining tolerance to self-antigen (6). However, when the signaling cascades initi-ated by signals 2 and 3 are disrupted it can lead to tolerance to pathogens, which leaves the host immunocompromised.

Because both signals 1 and 2 involve cell-cell interaction, it was important to con-firm these processes were functional under

Sejiro Littleton and Dylan Ludwick

2

30

microgravity conditions. In a series of experi-ments performed onboard MASER flights 3 and 4, as well as the MAXUS 2 flight, the binding of mitogen Con A to glycoproteins on the cell membrane was used as a mimic of signal 1 in order to assess the effects of microgravity on T cell functionality (7, 8, 3). Although the patching of Con A glycopro-teins after binding is significantly inhibited in microgravity in comparison to 1g-onboard and ground centrifuge controls, researchers concluded that the overall influence in such a rapid process was small, and unlikely to ac-count for the documented large scale inhibi-tion of T cell activation (9). In another exper-iment human peripheral blood lymphocytes, which include both T cells and APCs, formed progressively larger aggregate clusters 12-78 hours after stimulation with Con A indicating that physical contact between cells can occur under microgravity conditions (10).

Experiments modeling microgravity in the Random Positioning Machine (RPM) have found a significant dysfunction in the tran-scription of IL-2 and IL-2R alpha genes in lymphocytes stimulated with Con A (9). This confirms previous reports of decreased IL-2 and IL-2R in peripheral blood lymphocytes exposed to Con A in real microgravity on board Spacelab SLS-1 (4). As IL-2R and IL-2 production are the primary targets of signal 1 and 2 signaling cascades, their decrease could indicate incomplete or inefficient func-tion of signals 1 and 2. Regardless of the cause-and-effect relationship, the outcome is interference with signal 3 followed by T cell inactivation as a result of gravity unloading.

Comparisons of peripheral leukocyte sub-sets in crewmembers after short and long duration spaceflight missions have been per-formed, revealing time spent in microgravity to be an important factor in the analysis of immune adaptation to spaceflight (11). CD69 cell surface expression, a marker of early T cell activation, was elevated after stimulation with mitogens in short duration shuttle crew-members after landing, however production of Interferon-gamma (IFN-γ) was decreased. In contrast, long duration ISS crewmembers

displayed significant reduction in CD69 ex-pression, and unchanged production of IFN-γ. Both groups showed a decrease in IL-2 pro-duction, as well as a decrease in INF- γ:IL-10 ratio, suggesting a detectable T-helper type 2 (Th2) cytokine shift (11).

To start revealing the molecular mecha-nism behind the dysregulation of T cell function, experiments on board the ISS in-vestigated patterns of early gene expression between microgravity exposed T cells and 1g controls after 1.5 and 4 hours of stimulation with Con A and anti-CD28 (12). Researchers found 47 significantly down-regulated genes in the microgravity group at the 1.5 hour mark, including down-regulated key imme-diate early genes and their targets, such as transcription factors Rel/NF-kB, CREB, and Serum Response Factor (SRF). This confirms earlier reports of NF-kB and CREB pathway dysregulation, which was attributed to down-regulation of protein kinase A but not protein kinase C (13). The transcription of cREL and its gene targets were significantly reduced as well, and downstream Tumor Necrosis Factor (TNF) effector functions were identified as particularly gravity sensitive after genetic pathway analysis (12). Induction of NF-kB is a result of the costimulatory signals 2 and 3, and in the absence of this signaling cascade the cell can become anergic. Therefore, the alterations observed in the gene expression of T cells activated in microgravity is concern-ing because it might lead to immunosuppres-sion in astronauts. CREB plays an important role in the production of IL-2, IFN-γ, and the induction of T cell proliferation (14). The dysregulation of this pathway might there-fore explain the reduced IL-2 levels and the Th2 cytokine shift observed in previous ex-periments. Even with a transcriptionally per-missive methylation of the IFN-γ promoter, if CREB activity is reduced the production of IFN-γ might not be sufficient to promote a Th1 response, resulting in a cytokine shift and immunosuppression (15). Analysis at 4 hours identified 98 other genes down-regulated in microgravity, illustrating the interaction be-tween primary and secondary microgravity


3

31

effects (12). Authors note in particular that

SRF binding sites are present in the promot-

ers of a majority of genes inhibited in T cells

activated in microgravity (12). Given that

Ras/MAPK and Rho/actin pathways activate

SRF, these cytoskeleton-regulating small

GTPases could be disrupted in microgravity

explaining the reduced T cell activation (16).

If cytoskeletal regulatory elements are

impaired and lead to alterations in T cell ac-

tivation, then cell motility becomes an im-

portant consideration as the cytoskeleton is

the primary effector of this process. In vivo,

APCs must migrate to the lymph nodes in

order to present antigen to T cells (signal

1) and deliver a costimulatory signal 2 like-

wise T cells must enter lymph nodes through

the processes of extravasation. Should cells

lack normal motility then the immune sys-

tem could be impaired. In both the presence

and absence of mitogen stimulation, cells in

single suspension were shown to be signifi-

cantly more motile than 1g ground controls.

This may not hold true for all cell types, as

J-111 human monocytes exposed to 24 hours

of microgravity aboard the ISS displayed a

substantial reduction in motility in compari-

son to onboard 1g and ground controls (17).

Interestingly, a significant difference in mo-

tility was also noted between controls, most

likely due to the fact that inflight 1g samples were exposed to microgravity during ascent

and transfer to centrifuge. These results re-

inforce earlier studies in J-111 monocytes

exposed to 24 hours simulated microgravity

in the RPM, which showed similar impair-

ments in cell motility (18). It is conceivable

that diminished motility in monocytes or

other APCs may be substantial enough to in-

terfere with proper migration into lymphoid

organs, leading to decreased stimulation and

increased susceptibility to pathogens a po-

tential source for the observed latent viral

reactivation and symptoms of pathogenic

infection.

B Lymphocytes

Experiments investigating the humoral re-

sponse and the effects of microgravity on the

B cell populations are more limited than their

T cell counterparts (see Table 1 for compari-

son. Fortunately, antibody binding is not dis-

rupted in microgravity and Enzyme-Linked

Immunosorbent Assay (ELISA) tests can be

conducted aboard the ISS (19). Serum levels


Cell type Effects References

T lymphocytes

● disruption of activation (long term exposure)

● increase in activation (short term exposure), short-term decrease in INF-ɤ, decrease in INF-ɤ:IL-10 ratio

● increase in motility ● decrease in transcription

of IL-2 and IL-2R alpha

● down-regulation of 47 early gene targets, including Rel/NF-kB, CREB, SRF, PKA, and cREL

Konstantinova et al 1993 (2) , Cogoli et al 1988 (3), Cogoli et al

1993 (4), Pipia et al 1996 (5)

Crucian et al 2008 (11)

Cogoli-Greuter et al 1996 (10) Cogoli-Greuter 2004 (9)

Chang et al 2012 (12), Boonyaratanakronkit et al 2005

(13)

B lymphocytes ● some increase in IgM and IgA levels

Rykova et al 2008 (21), Konstantinova et al 1993 (2)

Table 1. Summary of the Relation between Microgravity and the Adaptive Immune System Table 1. Summary of the Relation between Microgravity and the Adaptive Immune System

4

32

headed by Namni Goel should help to es-tablish risk factors, useful biomarkers, and a standard for comparison when measuring the effects of stress. These results indicate that humoral immune response is not likely to be a prime risk factor for exploration class mis-sions, such as a Mars mission, however any unexpected complications that arise could be untreatable and place the crew and mission at severe risk (1).

INNATE IMMUNITY

Natural Killer Cells

NK cells comprise the third lymphocyte lineage and play an important role in the defense of the host against viruses and intra-cellular bacteria (28). These cells are named “natural killer” because unlike the other cells of the lymphocyte lineage they require no APC for activation. NK cells are able to recognize ligands on the surface of infected cells, stressed cells, and those lacking self-peptides presented on MHC-I to quickly identify threats and respond by lysing target cells as a primary defense against invaders (28). This innate defense provided by the NK cell allows time for the adaptive immune response to clonally expand and respond to the infection should it not be eradicated by the innate immune response (28). With the observed viral reactivation among many as-tronauts and the unknown virulence of many bacterial strains in microgravity, it becomes a priority to determine the ability of NK cells to maintain functional cytolytic activity.

Data analysis of NK cells extracted from the peripheral blood of 88 cosmonauts after returning from long term missions revealed two phenotypes. The first of these phenotypes was characterized by a static or increased percentage of CD3-/CD56+/CD16+ lympho-cytes compared to preflight, with reduced cytotoxic activity of the NK population. The second phenotype demonstrated significantly reduced fraction of CD3-/CD56+/CD16+ lymphocytes but significantly higher NK cell cytotoxic activity (29). A previous study on

of the immunoglobulin isotypes taken from many astronauts suggest little clinical risk of a dysfunctional humoral response of the im-mune system. Given no change to antibody binding in microgravity and no significant change in serum levels for each of the im-munoglobulins IgG, IgM, IgA, IgD, & IgE the risk in short term missions lasting around ten days is negligible (2, 20, 21).

It is less clear what the clinical risks are for longer missions such as those lasting 125 days or longer. The trend seems to indicate a maintenance of Ig within standard levels; however, cases have been documented where cosmonauts experienced an increase in both their IgM and IgA levels during long term flight (2, 21). Interestingly after 5 months in space, the newt, Pleurodeles waltl, one of the few vertebrate animals to be immunized in space, demonstrated a 3.5 fold increase in IgY heavy chain transcription, the equivalent of human IgA (22). It is unclear whether mi-crogravity is the direct factor responsible for this as the researchers were unable to con-trol for radiation and stress responses (22). However, the ability to class switch to IgY is consistent with the described Th2 cytokine shift.

Valeri Vladimirovich Polyakov holds the current record for longest spaceflight, a consecutive 438 days (23). No major com-plications due to altered immune response developed during his time in space. Viral reactivation has been observed in a number of subjects; however, this has not led to any significant health threat (24). It is possible this reactivation can be attributed to stress induced suppression of the cell mediated branch of the immune system as tests con-ducted in the Arctic Winter Over missions and the Haughton Mars Project (conducted on Devon Island in the Canadian high arc-tic) demonstrated similar viral reactivation with reduced T cell activity and function (1, 25, 26, 27). The Human Research Roadmap tasks “Individualized Stress Detection System” headed by Daniel Mollicone and “Biomarkers as Predictors of Resiliency and Susceptibility to Stress in Spaceflight”


5

33

ISS crewmembers and shuttle members, long and short duration missions respectively, found a significant reduction in NK cell numbers which persisted at least three days post flight, unfortunately no functional ac-tivity was measured in this experiment (11). Further studies should seek to understand the mechanism behind these two distinct pheno-types. Below we discuss some in vitro studies that addressed these issues.

Initial studies using two-dimensional (2D) clinostat and RPM seemed to indicate that simulated microgravity had no effect on the cytolytic activity of NK cells (30, 31). This would seem to suggest that the primary cause of the changes seen in human NK cells from peripheral blood of astronauts is more likely due to a combination of radiation and stress responses. However, these initial studies of-ten only tested incubation periods up to 24 hours. When the time was extended to 48 hours there was a significant reduction in the ability of NK cells to lyse target MHC-negative K562 cells and this inhibition was even more significant at 72 hours (31). When human NK cells were incubated under these same conditions, expression of IFN-γ, perfo-rin, and the receptors NKG2D and NKG2A were significantly reduced after 48 hours (31). Additionally human NK cells exposed to simulated microgravity for 48 hours were found to have an increased rate of early and late apoptosis in comparison with 1g and vertical rotational controls after Annexin V/Propidium Iodide flow cytometry (31). In contrast, Granzyme-B levels and the recep-tors NKp30 and NKp44 were unaffected (31). This decrease in receptor, IFN-γ and perforin levels and the increased rate of apoptosis is indicative of a lowered activity state of the NK population, and might explain the reduc-tion in cytolytic ability after incubation in simulated microgravity. Further experimen-tation revealed IL-15 alone or in combination with IL-12 can rescue this suppressed cyto-toxicity (31).

Despite these developments it still remains unclear to what extent the changes observed in the NK cell population of astronauts are

specifically due to each of the factors of mi-crogravity, stress, and radiation. However, should the risk of reduced NK cell function be sufficient for clinical intervention the ad-ministration of IL-12 and IL-15 presents as one possible solution (31).

Monocytes & Macrophages

Precursors to macrophages and dendritic cells, monocytes are large, granular, mono-nuclear cells that circulate the blood until tis-sue damage or inflammatory signals recruit them to extravasate and differentiate into ef-fector progeny (32). Macrophages are tissue resident and long lasting, they engulf patho-gens to present processed peptides on their surfaces which can be recognized to activate the adaptive immune system (28). The gran-ules present in these cells can also be used to activate oxidative burst reactions to disrupt and destroy pathogens (28). It is essential that the processes involved in cell motility and signaling remain unimpaired for a functional innate immune system, unfortunately results seem to indicate microgravity significantly impairs both of these aspects of monocyte and macrophage function.

Monocytes obtained from blood sam-ples of healthy volunteers were activated with LPS containing solution at preset time points, at 1g and 0gs; however, only 31 of 96 planned samples were suited for analysis due to technical failures (33). Results obtained indicated an increasing trend but no signifi-cant change in p38 MAP Kinase activity, as determined by the differences in mean fluo-rescent intensity, between experimental and control conditions (33). In contrast Jun-N-terminal kinase (JNK) activation is signifi-cantly reduced under spaceflight micrograv-ity conditions, though no changes in nuclear localization were noted (33). Interestingly, coactivation of p38 MAPK and JNK by in-flammatory signals occurs through distinct upstream kinases, MKK3/6 and MKK4/7 respectively which could explain the differ-ential effect of microgravity on these kinases (33). ERK, extracellular regulated kinase,


6

34

was also inhibited by microgravity albeit to a lesser degree than JNK, this was corroborated by results from another study which showed that microgravity affected phosphorylation in Jurkat T cells (33, 34). One should note the p38 MAPK pathway is implicated in early bacterial response whereas JNK has a role in the chronic inflammatory response and the mediation of Reactive Oxygen Species (ROS) to induce apoptosis and necrosis (33, 35). These results illustrate the differential effect of microgravity on kinases within cell populations and raises the question of what mechanism leads to these opposing effects.

Another important feature of macrophages affected by microgravity is their ability to initiate oxidative bursts in response to patho-gens. The production of ROS in NR8383 rat alveolar macrophages was shown to be sig-nificantly affected by changes in micrograv-ity, specifically reduced in microgravity and enhanced in hypergravity. These changes ap-pear to be both rapid and reversible within seconds (36). Studies were conducted in parabolic flight and 2D clinostats, thus de-spite the significance of the rapidly revers-ible changes, more studies are needed about the long term effects of microgravity expe-rienced during exploration class missions on tissue resident macrophages.

The steps in generation of ROS most likely to be gravity sensitive are those involved in the initial activation of a burst reaction. The spleen tyrosine kinase, Syk, plays an impor-tant role in the signaling pathway initiated by the binding of Pattern Recognition Receptors (PRRs) leading to oxidative burst and release of ROS. Phosphorylation of ITAMs by Src family kinases and recruitment and activation of Syk relies on adaptor proteins and occurs early in the pathway initiating oxidative burst (37, 38). Because the changes to ROS pro-duction are rapid and reversible, and down-stream NF-kB translocation and binding to DNA in the nucleus is largely unaffected, it is plausible that NF-kB can be compen-sated through some other signaling pathway, whereas the early signaling steps near the cell surface involving integrins and cytoskeletal


transport components for Syk phosphoryla-tion and ROS production are disrupted in microgravity (38). While not a cause in the experiments conducted in simulated micro-gravity, ionizing radiation has been shown to activate NF-kB through a Poly(ADP-ribose) Polymerase (PARP-1) (39).

An outcome of macrophage activation is the production of TNF-α and other pro-in-flammatory cytokines, yet another function that has been reported to be altered by mi-crogravity. Mouse macrophages stimulated with LPS and exposed to simulated gravity via Rotary Cell Culture System-1 (RCCS-1) for 24 hours resulted in significant depression TNF-α expression (40). Consistent with the results of Brungs et al., no effect was seen on translocation or DNA binding of NF-kB; however, no differences were observed in JNK or IKK phosphorylation. Researchers did note that heat shock factor 1 was acti-vated under these conditions and it is possible that stress responses could offer an alternate explanation to the reduced TNF-α expression (40).

Results from experiments analyzing the Toll-Like Receptors (TLR) of 14 ISS mem-bers demonstrate opposing and variable trends but also implicate heat shock factors in immune system shifts (29). In five cosmo-nauts the relative number TLR-expressing monocytes and neutrophilic granulocytes in peripheral blood on the 1st day after return from spaceflight was found to be signifi-cantly increased. In the remaining nine cos-monauts the opposite trend was observed by decreased representation of these cells. An increase in heat shock proteins was shown to be significant on the first day of landing. Ligands of the TLRs 2 and 4, these proteins are known to undergo significant increase in production and release from necrotic cells when an organism is exposed to biological, psychological or physical stressors, all of which an astronaut is known to experience (41). This protein production could have a protective effect on the innate immune cells expressing the proper receptors (29). The loss of bone density, thereby bone marrow and

7

35

the production of hematopoietic cells, might explain this decreases in the monocyte and neutrophil repertoire in the second group of cosmonauts (29).

Another study by Chongzen Wang further explored the changes induced by micrograv-ity in mouse macrophages by incubation with IL-4 in a RCCS-1 for 24 hours. They noted induction of the p38 MAPK pathway activ-ity downstream of the transcription factor C/EBPβ and significantly enhanced levels of arginase mRNA and protein (42). This in-crease of MAPK activity was confirmed in monocytes as well (33). Consistent with the maintenance of a Th2 shift observed in other experiments IL-6 production increased and IL-12B was depressed (11, 42).

To determine the effect of microgravity on macrophage differentiation one study analyzed the transcriptional profiles of mac-rophages derived from bone marrow of C57BL/6 mice after 14 days differentiation in the presence of rmM-CSF (recombinant mouse Macrophage Colony Stimulating Factor) (43). Phenotypically a significant increase of activated macrophages and in-creased expression levels of Mac2 and c-Fms markers were found in the space flight sample in comparison to the ground control. Researchers interpreted this finding as an in-dicator of a population of macrophages that is alternatively differentiated after spaceflight but not necessarily inhibited. Microarray analysis from this experiment found genes associated with the coagulation system, Fc-gamma receptor-mediated phagocytosis, endoplasmic reticulum stress and growth hormone signaling pathways to be most sig-nificantly reduced under the effects of micro-gravity in comparison to the ground control. Specifically, CCR5 and the kinases mTOR, p38 MAPK and FLT3 were significantly re-duced (43).

A summary of the innate immune system and the alterations that occur under the ef-fects of microgravity in NK cells, monocytes and macrophages are included in Table 2.

THE CYTOSKELETON

The cytoskeleton is an active and dy-namic intracellular network composed of three main types of polymer: actin filaments, microtubules, and intermediate filaments. Collectively, these proteins are responsible for the structural organization of cellular contents in space, physical and biochemical connections to the external environment, as well as generating the forces and contractions required for movement and change of shape (44). Important cellular processes such as signal transduction, growth, gene expression, and proliferation are all influenced by cyto-skeletal organization (45). Evidence mount-ing over the years suggests that the cytoskel-eton plays a key role in sensing changes in gravity as well (46, see Table 3, Figure 1).

In order for gravity to influence the cell, a mechanical signal must be converted to a chemical signal in a process known as mechanotransduction. Mediating this con-version are mechanosensitive structures, several of which have been associated with the cytoskeleton (47). Focal adhesions, sites of attachment between actin stress fibers and the cellular membrane, are known to house tension sensitive integrins and vinculin (48). Cadherins prominent in cellular junctions show mechanosensitivity as well. Even actin filaments themselves have been shown to be tension sensitive (49). In searching for the initial mechanism(s) of gravity sensing, the cytoskeleton and its associated elements ap-pear to be the most likely candidates.

Changes in the cytoskeleton in response to gravity unloading are widely reported for all three types of filaments amongst many differ-ent cell types (50, see Table 3) Human J-111 monocytes exposed to real microgravity aboard the ISS showed a significant decrease in the density of filamentous biopolymers of F-actin, with filaments localizing close to the plasma membrane as opposed to being orga-nized within the complex cytosolic network (17). This reinforces earlier observations of identical changes seen in cells undergoing simulated microgravity in the RPM (18).


8

36

Cell type Effects References

Natural killer cells ● phenotypic differentiation featuring both reduced and enhanced functional activity levels

● reduction in cell numbers (long term exposure)

● decrease in expression of IFN-y, perforin, and the receptors NKG2D and NKG2A

Rykova 2013 (29)

Crucian et al 2008 (11)

Li et al 2013 (31)

Monocytes

● reduction in motility ● disruption in Jun-N-

terminal kinase activation

● activation of p38 MAPK pathway

Meloni et al 2011 (17), 2006 (18)

Verhaar et al 2014 (33)

Macrophages

● rapid and reversible reduction in ROS production

● decrease in Syk phosphorylation

● depression of TNF-α

expression, activation of heat shock factor 1

● Activation of p38 MAPK

pathway, increased IL-6 production, decreased IL-12B production

Adrian et al 2013 (36)

Brungs et al 2015 (38)

Wang et al 2014 (40)

Wang et al 2015 (42)

Table 2. Summary of the Relation between Microgravity and the Innate Immune System Table 2. Summary of the Relation between Microgravity and the Innate Immune System

Actin filaments in osteoblasts experiencing 4 days of microgravity aboard the space trans-port system STS-56 shuttle flight also dem-onstrated notable disorganization, as well as a reduction in the number of stress fibers (51).

Lewis et al. first reported disruptions of the microtubule organizing center in nonadherent Jurkat cells within four hours of micrograv-ity (52). These changes reverted within 48 hours. Researchers noted that this effect was also seen in the on-board 1g control, and thus attributed it to vibrations from launch as op-posed to a real microgravity effect. Human T lymphocytes flown on MASER-12 suborbital

space flight mission, however, demonstrated significantly increased beta-tubulin expres-sion during the microgravity phase, which did not occur in the on-board 1g control (34). Meloni’s experiment with J-111 cells in real microgravity revealed highly disorganized b-tubulin architecture, with thickening in a perinuclear position which was not seen in on-board 1g or ground controls as well (16). Additionally, self-organization of microtu-bules in vitro largely failed to occur in sam-ples exposed to 13 minutes of weightlessness (53).

Studies focusing on intermediate filaments


9

37

Cytoskeletal Component

Effects Cell Types References

Actin ● decrease in density of F-actin filaments

● disorganization of actin filaments

● reduction in number of actin stress fibers

J-111 monocytes

Osteoblasts

Meloni et al 2011 (17), Crucian et al 2008 (11)

Hughes-Fulford and Lewis 1996

(51)

Tubulin ● disorganization of b-tubulin architecture

● increase in b-tubulin expression

J-111 monocytes

T lymphocytes


Tauber et al 2013 (34)

Intermediate Filaments

● thickening of vinculin, reorganized parallel to cell membrane

● reorganization of vimentin into thick bundles

● increase in integrin protein

subunit expression

● increase in Fibronectin and E-cadherin mRNA levels

J-111 monocytes

Jurkat cells, Breast cancer cells

Mesenchymal stem

cells

WB-F344 cells


Cogoli et al 1988 (3), Masiello et al 2014 (54)

Meyers et al 2004 (56)

Qu et al 2006 (57)

Functional Structures

● disorganization of complex cytosolic network

● reduction in number and size of focal adhesions

● reduction in number size of focal adhesions

● reversible disruption of

microtubule organizing center

● reduction in

phosphorylation of focal adhesion-associated kinases

J-111 monocytes

Osteoblasts

Jurkat cells

Mesenchymal stem cells


Guignandon et al 1997 (55)

Lewis et al 1998 (52)

Meyers et al 2004 (56)

Table 3. Summary of the Relation between Microgravity and the Cytoskeleton

Table 3. Summary of the Relation between Microgravity and the Cytoskeleton

are limited, however an experiment per-formed with Jurkat cells in real microgravity aboard MAXUS 2 recorded a striking reorga-nization of the intermediate filament vimen-tin into thick bundles within 30 seconds of exposure, in comparison to the thin networks seen in Earth gravity controls (3). Breast can-cer cells undergoing simulated microgravity in the RPM showed similar rearrangement of vimentin (54). Interestingly, these cells di-verged into two nearly equal phenotypes of adherent cells and free floating cell clumps, each with disorganized but notably different

cytoskeletal rearrangements. Mechanosensitive structures associated

with the cytoskeleton are likely key compo-nents in cellular adaptation to microgravity, however more work needs to be done in order to fully understand the process and players involved. Meloni’s J-111 monocytes dem-onstrated thickening of the focal adhesion anchor protein vinculin in real microgravity, which reorganized parallel to the cell mem-brane (17). The size and number of focal ad-hesions per cell was also reduced, in accor-dance with the reduction of actin stress fibers.


10

38


Figure 1. A working model of the cytoskeleton at zero gravity and its relation to immune dysfunction.

Figure 1. A working model of the cytoskeleton at zero gravity and its relationship to immune dysfunction. Alterations to the cytoskeleton cause disruption to a variety of cell functions in-cluding motility, signal transduction, cell proliferation and gene expression. Due to the require-ment of immune cells to hone to a site of infection and respond according to specific stimulus it is suggested the changes in the cytoskeleton lead to impaired immune function in zero gravity environments.

11

39

This is consistent with earlier parabolic flight data regarding similar changes in osteoblast focal adhesions (55). Human mesenchymal stem cells cultured for 7 days in simulated microgravity showed increases in integrin protein subunit expression, but significant reduction in phosphorylation of downstream focal adhesion-associated kinases (56).

Additionally, rat WB-F344 cells under simulated microgravity displayed signifi-cantly increased levels of Fibronectin and E-cadherin mRNA (57). These effects may relate to observations by Levenberg et al., which revealed an autoregulatory system whereby stimulation through cadherins or integrins specifically and preferentially en-hanced the formation of its associated adhe-sive structure. Essentially, when cell-cell ad-herence junctions are increased, cell-matrix focal adhesions are decreased and vice versa (58). This dichotomy could be a possible source of variation seen in cellular adaptation to microgravity, such as the dual cytoskeletal phenotypes seen in the breast cancer cell line, or the motility differences between T lym-phocytes and monocytes.

Finally, a recent review paper by Louis et al. described how nearly all of the cytoskel-etal changes associated with microgravity could be attributed to the molecular workings of Rho GTPases. The authors hypothesized that cells exposed to microgravity respond with depressed RhoA activity and increased Rac1 activity initially, which changes to de-pressed activity of both proteins in the long term (59). Additionally, Guignandon et al. have found that silencing of Rho GTPases Rac1 and the protein Cdc42, but not RhoA, effectively counteracted the reduction in number of focal adhesions and F-actin fibers seen in osteoblasts exposed to real micro-gravity (60). Although the initial mechanisms of gravity sensing have yet to be fully illu-minated, it appears that we may be one step closer to the source.

CONCLUSION

We have come a long way from our initial

observations of spaceflight-associated im-mune dysfunction. Simulated low gravity techniques in the 2D-clinostat and RPM have allowed for detailed comparison to more complicated studies in real microgravity aboard parabolic flights or the ISS. Decades of research into the effects of microgravity suggest a dynamic cellular adaptation process composed of primary and secondary down-stream responses. The cytoskeleton holds a substantial influence over signal transduc-tion, gene expression, and proliferation, and its early disruption seems to precede many of the observed changes in both adaptive and in-nate immunity (see Figure 1). That being said, the picture is far from clear. An exact mecha-nism of gravity sensing remains obscure. The notable variations in results reflects the expo-sure-dependent nature of this microgravity-mediated process, as well as the wide-scale variances in experimental methodology. Short timescales must be used in searching for the presence of an initial gravity sensor, while longer timescales will be useful in dis-tinguishing between short term and long term adaptational effects. RhoGTPases appear to be a promising candidate for upstream, grav-ity-sensitive regulators of the cytoskeleton, and Louis et al. further predict the role of GAP (GTPase Activating Protein) and GEF (Guanine Exchange Factor) RhoGTPase regulators (inhibitors and activators, respec-tively) in this cellular adaptation process (59). Future experiments in real micrograv-ity which monitor these specific activities at onset of exposure will be a critical next step in developing our understanding of gravity-sensing by non-specialized cells.

While the disruptive impacts of micrograv-ity on the immune system are apparent, more work needs to be done in order to determine the relative risk it poses for astronauts engag-ing in extended spaceflight. Clinical studies assessing immune functioning of astronauts at regular intervals during and after longer duration missions aboard the ISS will be helpful in this regard. An ongoing research mission, the NASA Twins Study, is working to address some of these issues by comparing


12

40

real and simulated low gravity conditions demonstrated altered gene expression and enhanced virulence (62). Far more research in both real and simulated microgravity will be necessary to properly address the potential effects of microgravity on microbial patho-genicity. With factors such as stress, sleep deprivation, as well as solar and cosmic ra-diation largely unaccounted for, microgravity will be but one of many potential hurdles as humankind pushes ever deeper into the beck-oning cosmos.

the physiological changes that occur over the course of one year spent on the ISS (61). The researchers will compare complete genome sequencing, metabolic markers, and a variety of other data generated from experiments on Mikhail Kornienko and Scott Kelly during their time in microgravity with Scott’s mono-zygotic twin brother retired astronaut Mark Kelly who will remain on Earth as a control.

At least one study suggests that micrograv-ity may affect pathogens as well, as bacte-rium Salmonella typhimurium grown in both


REFERENCES

1. Crucian, B., R. P. Stowe, M. C. Ott, J. L. Becker, R. Haddon, K. A. McMonigal, and C. F. Sams. 2009. Risk of Crew Adverse Health Event Due to Altered Immune Response.

2. Konstantinova, I. V., M. P. Rykova, A. T. Lesnyak, and E. A. Antropova. 1993. Immune changes during long-duration missions. J Leukoc Biol. 54: 189-201.

3. Cogoli, A., B. Bechler, O. Muller, and E. Hunzinger. 1988. Effect of microgravity on lym-phocyte activation. In Biorack on Spacelab D1. ESA Publication Division, ESTEC Noordwijk 89-100.

4. Cogoli, A., B. Bechler, M. Cogoli-Greuter, S. B. Criswell, H. Joller, P. Joller, E. Hunzinger, and O. Müller. 1993. Mitogenic signal transduction in T lymphocytes in microgravity. J Leukoc Biol. 53: 569-575.

5. Pippia, P., L. Sciola, M. Cogoli-Greuter, M. A. Meloni, A. Spano, and A. Cogoli. 1996. Activation signals of T lymphocytes in microgravity. J. Biotechnol. 47: 215-222.

6. Harris, N. I. and F. Ronchese. 1999. The Role of B7 costimulation in T-cell Immunity. Immunol Cell Biol 77: 304-311.

7. Cogoli, M., B. Bechler, A. Cogoli, N. Arena, S. Barni, P. Pippia, G. Sechi, N. Valora, and R. Monti. 1990. Lymphocytes on Sounding Rockets. Proceedings of the 4th European Symposium on Life Sciences Research in Space, Trieste 229-234.

8. Cogoli, M., B. Bechler, A. Cogoli, N. Arena, S. Barni, P. Pippia, G. Sechi, N. Valora, and R. Monti. 1992. Lymphocytes on sounding rockets. Adv. Space Res. 12: 141-144.

9. Cogoli-Greuter, M. 2004. Effect of gravity changes on the cytoskeleton in human lympho-cytes. Gravity and Space Bio Bulletin 17: 27.

13

41


10. Cogoli-Greuter, M., M. A. Meloni, L. Sciola, A. Spano, P. Pippia, G. Monaco, and A. Cogoli. 1996. Movements and interactions of leukocytes in microgravity. J. Biotechnol. 47: 279-287.

11. Crucian, B. E., R. P. Stowe, D. L. Pierson, and C. F. Sams. 2008. Immune System Dysregulation Following Short- vs Long- Duration Spaceflight. Aviat Space Environ Med 79: 835.

12. Chang, T. T., I. Walther, C. F. Li, J. B. Boonyaratanakornkit, G. Galleri, M. A. Meloni, P. Pippia, A. Cogoli, and M. Hughes-Fulford. 2012. The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol. 92: 1133-1145.

13. Boonyaratanakornkit, J. B., A. Cogoli, C. F. Li, T. Schopper, P. Pippia, G. Galleri, M. A. Meloni, and M. Hughes-Fulford. 2005. Key gravity-sensitive signaling pathways drive T cell activation. Faseb j. 19: 2020-2022.

14. Wen, A.Y., Sakamoto, K.M., and Miller, L.S. 2010. The role of the transcription factor CREB in immune function. J Immunol 185: 6413–6419.

15. Yano, S., Ghosh, P., Kusaba, H., Buchholz, M., and Longo, D.L. 2003. Effect of promoter methylation on the regulation of IFN-gamma gene during in vitro differentiation of human pe-ripheral blood T cells into a Th2 population. J Immunol 171: 2510–2516.

16. Katsch, K., de Jong, S.J., Albrecht, J.-C., Steger, J., Genth, H., Posern, G., and Biesinger, B. 2012. Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip. Cell Communication and Signaling : CCS 10: 5–5.

17. Meloni, M. A., G. Galleri, G. Pani, A. Saba, P. Pippia, and M. Cogoli-Greuter. 2011. Space Flight Affects Motility and Cytoskeletal Structures in Human Monocyte Cell Line J-111. Cytoskeleton (Hoboken) 68: 125.

18. Meloni, M. A., G. Galleri, P. Pippia, and M. Cogoli-Greuter. 2006. Cytoskeleton changes and impaired motility of monocytes at modelled low gravity. Protoplasma 229: 243-249.

19. Maule, J., M. Fogel, A. Steele, N. Wainwright, D. L. Pierson, and D. S. McKay. 2003. Antibody binding in altered gravity: implications for immunosorbent assay during space flight. J. Gravit Physiol. 10: 47-55.

20. Voss, E. W. 1984. Prolonged weightlessness and humoral immunity. Science 225: 214-215.

21. Rykova, M. P., E. N. Antropova, I. M. Larina, and B. V. Morukov. 2008. Humoral and cel-lular immunity in cosmonauts after the ISS missions. Acta Astronaut. 63: 697-705.

22. Boxio, R., C. Dournon, and J. P. Frippiat. 2005. Effects of a long-term spaceflight on immu-noglobulin heavy chains of the urodele amphibian Pleurodeles waltl. J. Appl. Physiol. (1985) 98: 905-910.

14

42

23. Siddiqi, A.A. (2015). Valeri Vladimirovich Polyakov.

24. Mehta, S. K., D. L. Pierson, H. Cooley, R. Dubow, and D. Lugg. 2000. Epstein-Barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners. J. Med. Virol. 61: 235-240.

25. Crucian, B., P. Lee, R. Stowe, J. Jones, R. Effenhauser, R. Widen, and C. Sams. 2007. Immune system changes during simulated planetary exploration on Devon Island, high arctic. BMC Immunology 8: 7.

26. Mehta, S. K., R. P. Stowe, A. H. Feiveson, S. K. Tyring, and D. L. Pierson. 2000. Reactivation and shedding of cytomegalovirus in astronauts during spaceflight. J. Infect. Dis. 182: 1761-1764.

27. Tingate, T. R., D. J. Lugg, H. K. Muller, R. P. Stowe, and D. L. Pierson. 1997. Antarctic isolation: immune and viral studies. Immunol. Cell Biol. 75: 275-283.

28. Abbas, A. K., A. H. H. Lichtman, and S. Pillai. 2011. Cellular and Molecular Immunology: with STUDENT CONSULT Online Access. Elsevier Health Sciences.

29. Rykova, M. P. 2013. Immune system in Russian cosmonauts after orbital space flights. Fiziol. Cheloveka 39: 126-136.

30. Buravkova, L. B., M. P. Rykova, V. Grigorieva, and E. N. Antropova. 2004. Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro. J Gravit Physiol 11: P177-180.

31. Li, Q., Q. Mei, T. Huyan, L. Xie, S. Che, H. Yang, M. Zhang, and Q. Huang. 2013. Effects of Simulated Microgravity on Primary Human NK cells. Astrobiology 13: 703.

32. Nichols, B. A., D. F. Bainton, and M. G. Farquhar. 1971. DIFFERENTIATION OF MONOCYTES : Origin, Nature, and Fate of Their Azurophil Granules. J. Cell Biol. 50: 498-515.

33. Verhaar, A. P., E. Hoekstra, A. S. W. Tjon, W. K. Utomo, J. J. Deuring, E. R. M. Bakker, V. Muncan, and M. P. Peppelenbosch. 2014. Dichotomal effect of space flight-associated micro-gravity on stress-activated protein kinase in innate immunity. Scientific Reports 4.

34. Tauber, S., S. Hauschild, C. Crescio, C. Secchi, K. Paulsen, A. Pantaleo, A. Saba, I. Buttron, C. S. Thiel, A. Cogoli, P. Pippia, and O. Ulrich. 2013. Signal transduction in primary human T lymphocytes in altered gravity - results of the MASER- 12 suborbital space flight mission. Cell Commun Signal 11: 32.

35. Shen, H. M. and Z. G. Liu. 0619. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radical Biology & Medicine JID - 8709159.

36. Adrian, A., K. Schoppmann, J. Sromicki, S. Brungs, M. von der Wiesche, B. Hock, W. Kolanus, R. Hemmersbach, and O. Ullrich. 2013. The oxidative burst reaction in mammalian cells depends on gravity. Cell Commun Signal 11: 98.


15

43


37. Hirose, M., J. Kitano, Y. Nakajima, K. Moriyoshi, S. Yanagi, H. Yamamura, T. Muto, H. Jingami, and S. Nakanishi. 2004. Phosphorylation and recruitment of Syk by immunorecep-tor tyrosine-based activation motif-based phosphorylation of tamalin. J. Biol. Chem. 279: 32308-32315.

38. Brungs, S., W. Kolanus, and R. Hemmersbach. 2015. Syk phosphorylation - a gravity sensi-tive step in macrophage signaling. Cell Commun Signal 13: 9.

39. Veuger, S. J., J. E. Hunter, and B. W. Durkacz. 2008. Ionizing radiation-induced NF-kappa]B activation requires PARP-1 function to confer radioresistance. Oncogene 28: 832-842.

40. Wang, C., H. Luo, L. Zhu, F. Yang, Z. Chu, H. Tian, M. Feng, Y. Zhao, and P. Shang. 2014. Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha expression in macrophage cells. Inflamm. Res. 63: 91-98.

41. Asea, A. 2008. Heat shock proteins and toll-like receptors. Handb Exp Pharmacol 183: 111–127.

42. Wang, C., H. Chen, H. Luo, L. Zhu, Y. Zhao, H. Tian, R. Wang, P. Shang, and Y. Zhao. 2015. Microgravity activates p38 MAPK-C/EBPÎ² pathway to regulate the expression of arginase and inflammatory cytokines in macrophages. Inflammation Res. 64: 303-311.

43. Ortega, M.T., Lu, N., and Chapes, S.K. 2012. Evaluation of in vitro macrophage differentia-tion during space flight. Adv Space Res 49.

44. Fletcher, D. A. and R. D. Mullins. 2010. Cell mechanics and the cytoskeleton. Nature 462: 485-492.

45. Cooper, G. M. 2000. Signal Transduction and the Cytoskeleton. In The Cell: A Molecular Approach, 2nd Edition ed. Sinauer Associates, Sunderland, MA.

46. Ingber, D. 1999. How cells (might) sense microgravity. Faseb j. 13: 3-15.

47. Ingber, D. 2006. Cellular mechanotransduction: putting all the pieces together again. Faseb j. 29: 811-827.

48. Monici, M. 2010. In Cell Mechanochemistry: Biological Systems and Factors Inducing Mechanical Stress, Such as Light, Pressure and Gravity, First Edition ed. M. Monici ed. Transworld Research Network, 75-96.

49. Galkin, V. E., A. Orlova, and E. H. Egelmanemail. 2012. Actin Filaments as Tension Sensors. Current Biology 22: R96-R101.

50. Vorselen, D., W. H. Roos, F. C. MacKintosh, G. J. L. Wuite, and J. J. van Loon. 2014. The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells. Faseb j 28: 536.

51. Hughes-Fulford, M. and M. L. Lewis. 1996. Effects of Microgravity on Osteoblast Growth Activation. Exp. Cell Res. 224: 103-109.

16

44

52. Lewis, M. L., J. L. Reynolds, L. A. Cubano, J. P. Hatton, B. D. Lawless, and E. H. Piepmeier. 1998. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat). Faseb j. 12: 1007-1018.

53. Papaseit, C., N. Pochon, and J. Tabony. 2000. Microtubule self-organization is gravity-dependent. Pnas 97: 8364-8368.

54. Masiello, M. G., A. Cucina, S. Proietti, A. Palombo, P. Coluccia, F. D’Anselmi, S. Dinicola, A. Pasqualato, V. Morini, and M. Bizzarri. 2014. Phenotypic Switch Induced by Simulated Microgravity on MDA-MB-231 Breast Cancer Cells. BioMed Research International 2014: 12.

55. Guignandon, A., Y. Usson, N. Laroche, M. H. Lafage-Proust, O. Sabido, C. Alexandre, and L. Vico. 1997. Effects of intermittent or continuous gravitational stresses on cell-matrix adhe-sion: quantitative analysis of focal contacts in osteoblastic ROS 17/2.8 cells. Exp. Cell Res. 236: 66-75.

56. Meyers, V. E., M. Zayzafoon, S. R. Gonda, W. E. Gathings, and J. M. McDonald. 2004. Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells. J Cell Biochem. 93: 697-707.

57. Qu, X. J., H. X. Li, S. D. Sun, and M. F. Feng. 2006. Three-dimensional spheroid model for cultivating WB-F344 cells in simulated microgravity. [Article in Chinese]. Sheng Wu Gong Cheng Xue Bao 22: 672-676.

58. Levenberg, S., B. Z. Katz, K. M. Yamada, and B. Geiger. 1998. Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands. J Cell Sci. 111: 347-357.

59. Louis, F., C. Deroanne, B. Nusgens, L. Vico, and A. Guignandon. 2015. RhoGTPasas as Key Players in Mammalian Cell Adaptation to Microgravity. Biomed Res Int. 2015: 17 pp.

60. Guignandon, A., C. Faure, T. Neutelings, A. Rattner, P. Mineur, M. T. Linossier, N. Laroche, C. Lambert, C. Deroanne, B. Nusgens, R. Demets, A. Colige, and L. Vico. 2014. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells. Faseb j 28: 4077-4087.

61. Gushanas, T. (July 30, 2015) NASA Twin Study [web site] Available from https://www.nasa.gov/twins-study.

62. Wilson, J. W., C. M. Ott, K. Höner zu Bentrup, R. Ramamurthy, L. Quick, S. Porwollik, P. Cheng, M. McClelland, G. Tsaprailis, T. Radabaugh, A. Hunt, D. Fernandez, E. Richter, M. Shah, M. Kilcoyne, L. Joshi, M. Nelman-Gonzalez, S. Hing, M. Parra, P. Dumars, K. Norwood, R. Bober, J. Devich, A. Ruggles, C. Goulart, M. Rupert, L. Stodieck, P. Stafford, L. Catella, M. J. Schurr, K. Buchanan, L. Morici, J. McCracken, P. Allen, C. Baker-Coleman, T. Hammond, J. Vogel, R. Nelson, D. L. Pierson, H. M. Stefanyshyn-Piper, and C. A. Nickerson. 2007. Space flight alters bacterial gene expression and virulence and reveals role for global regulator Hfq. Pnas 104: 16299-16304.


17

altered immune function in space: implications of a ... · well, and downstream tumor necrosis...

Documents